Efficient 2,3-Butanediol/Acetoin Production Using Whole-Cell Biocatalyst with a New Nadh/Nad(+) Regeneration System

An auto-inducing expression system was developed that could express target genes in S. marcescens MG1. Using this system, MG1 was constructed as a whole-cell biocatalyst to produce 2,3-butanediol/acetoin. Formate dehydrogenase (FDH) and 2,3-butanediol dehydrogenase were expressed together to build an NADH regeneration system to transform diacetyl to 2,3-butanediol. After fermentation, the extract of recombinant S. marcescens MG1ABC (pETDuet-bdhA-fdh) showed 2,3-BDH activity of 57.8 U/mg and FDH activity of 0.5 U/mg. And 27.95 g/L of 2,3-BD was achieved with a productivity of 4.66 g/Lh using engineered S. marcescens MG1(Pswnb+pETDuet-bdhA-fdh) after 6 h incubation. Next, to produce 2,3-butanediol from acetoin, NADH oxidase and 2,3-butanediol dehydrogenase from Bacillus subtilis were co-expressed to obtain a NAD+ regeneration system. After fermentation, the recombinant strain S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC) showed AR activity of 212.4 U/mg and NOX activity of 150.1 U/mg. We obtained 44.9 g/L of acetoin with a productivity of 3.74 g/Lh using S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC). This work confirmed that S. marcescens could be designed as a whole-cell biocatalyst for 2,3-butanediol and acetoin production.

Developing an Ethanol Utilization Pathway based NADH Regeneration System in Escherichia coli

Interests remain in searching for cofactor regeneration system with higher efficiency at lower substrate cost. Glucose dehydrogenase (GDH) system has been dominant in NADH regeneration, but it only has a theoretical yield of one NADH per glucose molecule. This work sought to explore the utility of a two-step ethanol utilization pathway (EUP) in pathway-based NADH regeneration. The pathway runs from ethanol to acetaldehyde and to acetyl-CoA with each step generating one NADH, that together results in a higher theoretical yield of two NADH per ethanol molecule. In this project, anaerobic biotransformation of ketone (acetophenone or butanone) to alcohol by cpsADH from Candida parapsilosis was used as readout for evaluating relative efficacy and operating modes for EUP cofactor regeneration in Escherichia coli BL21 (DE3). Experiment tests validated that EUP was more efficient than GDH in NADH regeneration. Further, growing cell delivered higher biotransformation efficiency compared to resting cell due to the driving force generated by cell growth. Finally, preculture or cultivation in M9 + 10 g/L ethanol medium delivered higher biotransformation efficiency compared to LB medium. Overall, EUP could help regenerate NADH in support of a biocatalytic reaction, and is more efficient in cofactor regeneration than GDH.

Genome-wide scans for signatures of selection in Mangalarga Marchador horses using high-throughput SNP genotyping

Background The detection of signatures of selection in genomic regions provides insights into the evolutionary process, enabling discoveries regarding complex phenotypic traits. In this research, we focused on identifying genomic regions affected by different selection pressures, mainly highlighting the recent positive selection, as well as understanding the candidate genes and functional pathways associated with the signatures of selection in the Mangalarga Marchador genome. Besides, we seek to direct the discussion about genes and traits of importance in this breed, especially traits related to the type and quality of gait, temperament, conformation, and locomotor system. Results Three different methods were used to search for signals of selection: Tajima’s D (TD), the integrated haplotype score (iHS), and runs of homozygosity (ROH). The samples were composed of males (n = 62) and females (n = 130) that were initially chosen considering well-defined phenotypes for gait: picada (n = 86) and batida (n = 106). All horses were genotyped using a 670 k Axiom® Equine Genotyping Array​ (Axiom MNEC670). In total, 27, 104 (chosen), and 38 candidate genes were observed within the signatures of selection identified in TD, iHS, and ROH analyses, respectively. The genes are acting in essential biological processes. The enrichment analysis highlighted the following functions: anterior/posterior pattern for the set of genes (GLI3, HOXC9, HOXC6, HOXC5, HOXC4, HOXC13, HOXC11, and HOXC10); limb morphogenesis, skeletal system, proximal/distal pattern formation, JUN kinase activity (CCL19 and MAP3K6); and muscle stretch response (MAPK14). Other candidate genes were associated with energy metabolism, bronchodilator response, NADH regeneration, reproduction, keratinization, and the immunological system. Conclusions Our findings revealed evidence of signatures of selection in the MM breed that encompass genes acting on athletic performance, limb development, and energy to muscle activity, with the particular involvement of the HOX family genes. The genome of MM is marked by recent positive selection. However, Tajima’s D and iHS results point also to the presence of balancing selection in specific regions of the genome.

Developing an ethanol utilisation pathway based NADH regeneration system in Escherichia coli

Many industrially relevant biotransformation in whole-cells are dependent on cofactors such as NADH or NADPH. Cofactor regeneration is an established approach for providing a cheap source of cofactors in support of the main biotransformation reaction in biocatalysis. In essence, cofactor regeneration uses a sacrificial substrate to help regenerate a cofactor consumed by the main biotransformation reaction. Enzymatic in nature, alternative cofactor regeneration systems with high efficiency and which utilises low cost sacrificial substrate are of interest. Glucose dehydrogenase system has been dominant in NADH regeneration. But, in its current incarnation, glucose dehydrogenase system is relatively inefficient in regenerating NADH with theoretical yield of one NADH per glucose molecule. This work sought to explore the utility of a two-gene ethanol utilisation pathway in NADH regeneration. Comprising the first step that takes ethanol to acetaldehyde, and a second step that converts acetaldehyde to acetyl-CoA, NADH from both steps could be mined for supporting biotransformation reaction in cofactor regeneration mode. Theoretically, ethanol utilisation pathway (EUP) affords a higher NADH yield of two NADH per ethanol molecule, and is therefore more efficient than glucose dehydrogenase (GDH) system. In this project, the EUP pathway was coupled to a cpsADH (an alcohol dehydrogenase from Candida parapsilosis) mediated ketone to alcohol anaerobic biotransformation with concentration of alcohol product as marker for efficiency of cofactor regeneration. Experiment tests showed that EUP was more efficient than GDH. Further, EUP could support biotransformation of both butanone and acetophenone in single and two-phase biotransformation, respectively. Additional work conducted to improve biotransformation efficiency revealed that ethanol provision positively correlated with biotransformation efficiency. Growing cell biotransformation was also found to improve biotransformation efficiency compared to resting cell due largely to the driving force generated by cell growth. Tests of a growth medium effect also found that cells cultivated in M9 ethanol medium delivered higher biotransformation efficiency compared to those cultivated in LB medium. This could arise due to the lower expression of NADH dependent enzymes during growth in M9 ethanol medium compared to LB medium that allowed more NADH to be diverted to support ketone biotransformation. However, a persistent problem with the experimental system is the relatively poor consumption of ethanol that points to need for further engineering of the system. Collectively, pathway-based NADH regeneration is possible with ethanol utilisation, with biotransformation efficiency dependent on mode of biotransformation (resting cell versus growing cell) and growth medium used.

The Effect of the Expression of the Antiapoptotic BHRF1 Gene on the Metabolic Behavior of a Hybridoma Cell Line

One of the most important limitations of mammalian cells-based bioprocesses, and particularly hybridoma cell lines, is the accelerated metabolism related to glucose and glutamine consumption. The high uptake rates of glucose and glutamine (i.e., the main sources of carbon, nitrogen and energy) lead to the production and accumulation of large amounts of lactate and ammonia in culture broth. Lactate and/or ammonia accumulation, together with the depletion of the main nutrients, are the major causes of apoptosis in hybridoma cell cultures. The KB26.5 hybridoma cell line, producing an IgG3, was engineered with BHRF1 (KB26.5-BHRF1), an Epstein–Barr virus-encoded early protein homologous to the antiapoptotic protein Bcl-2, with the aim of protecting the hybridoma cell line from apoptosis. Surprisingly, besides achieving effective protection from apoptosis, the expression of BHRF1 modified the metabolism of the hybridoma cell line. Cell physiology and metabolism analyses of the original KB26.5 and KB26.5-BHRF1 revealed an increase of cell growth rate, a reduction of glucose and glutamine consumption, as well as a decrease in lactate secretion in KB26.5-BHRF1 cells. A flux balance analysis allowed us to quantify the intracellular fluxes of both cell lines. The main metabolic differences were identified in glucose consumption and, consequently, the production of lactate. The lactate production flux was reduced by 60%, since the need for NADH regeneration in the cytoplasm decreased due to a more than 50% reduction in glucose uptake. In general terms, the BHRF1 engineered cell line showed a more efficient metabolism, with an increase in biomass volumetric productivity under identical culture conditions.

Recombinant Enzymatic Redox Systems for Preparation of Aroma Compounds by Biotransformation

The aim of this study was to develop immobilized enzyme systems that reduce carbonyl compounds to their corresponding alcohols. The demand for natural aromas and food additives has been constantly growing in recent years. However, it can no longer be met by extraction and isolation from natural materials. One way to increase the availability of natural aromas is to prepare them by the enzymatic transformation of suitable precursors. Recombinant enzymes are currently being used for this purpose. We investigated trans-2-hexenal bioreduction by recombinant Saccharomyces cerevisiae alcohol dehydrogenase (ScADH1) with simultaneous NADH regeneration by recombinant Candida boidinii formate dehydrogenase (FDH). In a laboratory bioreactor with two immobilized enzymes, 88% of the trans-2-hexenal was transformed to trans-2-hexenol. The initial substrate concentration was 3.7 mM. The aldehyde destabilized ScADH1 by eluting Zn2+ ions from the enzyme. A fed-batch operation was used and the trans-2-hexenal concentration was maintained at a low level to limit the negative effect of Zn2+ ion elution from the immobilized ScADH1. Another immobilized two-enzyme system was used to reduce acetophenone to (S)-1-phenylethanol. To this end, the recombinant alcohol dehydrogenase (RrADH) from Rhodococcus ruber was used. This biocatalytic system converted 61% of the acetophenone to (S)-1-phenylethanol. The initial substrate concentration was 8.3 mM. All enzymes were immobilized by poly-His tag to Ni2+, which formed strong but reversible bonds that enabled carrier reuse after the loss of enzyme activity.