You get what you screen for: on the value of fermentation characterization in high-throughput strain improvements in industrial settings.

While design and high-throughput build approaches in biotechnology have increasingly gained attention over the past decade, approaches to test strain performance in high-throughput have received less discussion in the literature. Here, we describe how fermentation characterization can be used to improve the overall efficiency of high-throughput DBTAL (design-build-test-analyze-learn) cycles in an industrial context. Fermentation characterization comprises an in-depth study of strain performance in a bioreactor setting and involves semi-frequent sampling and analytical measurement of substrates, cell densities and viabilities, and (by)products. We describe how fermentation characterization can be used to (1) improve (high-throughput) strain design approaches; (2) enable the development of bench-scale fermentation processes compatible with a wide diversity of strains; and (3) inform the development of high-throughput plate-based strain testing procedures for improved performance at larger scales.

Rapid and reliable DNA assembly via ligase cycling reaction.

Assembly of DNA parts into DNA constructs is a foundational technology in the emerging field of synthetic biology. An efficient DNA assembly method is particularly important for high-throughput, automated DNA assembly in biofabrication facilities and therefore we investigated one-step, scarless DNA assembly via ligase cycling reaction (LCR). LCR assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a thermostable ligase to join DNA backbones, and multiple denaturation-annealing-ligation temperature cycles to assemble complex DNA constructs. The efficiency of LCR assembly was improved ca. 4-fold using designed optimization experiments and response surface methodology. Under these optimized conditions, LCR enabled one-step assembly of up to 20 DNA parts and up to 20 kb DNA constructs with very few single-nucleotide polymorphisms (<1 per 25 kb) and insertions/deletions (<1 per 50 kb). Experimental comparison of various sequence-independent DNA assembly methods showed that circular polymerase extension cloning (CPEC) and Gibson isothermal assembly did not enable assembly of more than four DNA parts with more than 50% of clones being correct. Yeast homologous recombination and LCR both enabled reliable assembly of up to 12 DNA parts with 60-100% of individual clones being correct, but LCR assembly provides a much faster and easier workflow than yeast homologous recombination. LCR combines reliable assembly of many DNA parts via a cheap, rapid, and convenient workflow and thereby outperforms existing DNA assembly methods. LCR assembly is expected to become the method of choice for both manual and automated high-throughput assembly of DNA parts into DNA constructs.

Impact of dissolved hydrogen partial pressure on mixed culture fermentations.

Mixed culture fermentations are of interest for the low-cost production of organic acids from complex agricultural waste streams. Models are developed for these processes in order to predict the product spectrum as a function of the environmental process conditions. An important assumption in many existing models for anaerobic mixed culture fermentations is that the NADH/NAD(+) ratio is directly coupled to the dissolved hydrogen partial pressure (pH2, liquid). In this study, this assumption was tested experimentally with mixed culture chemostats operated at dilution rates of 0.05 and 0.125 h(-1) for a wide range of calculated dissolved hydrogen partial pressures (0.04-6.8 atm). No correlation was found between pH2, liquid and the NADH/NAD(+) ratio. This result, together with thermodynamic calculations, suggests that additional electron carriers such as ferredoxin and formate should be included in models predicting product formation by mixed cultures.

In vivo analysis of Saccharomyces cerevisiae plasma membrane ATPase Pma1p isoforms with increased in vitro H+/ATP stoichiometry.

Plasma membrane H(+)-ATPase isoforms with increased H(+)/ATP ratios represent a desirable asset in yeast metabolic engineering. In vivo proton coupling of two previously reported Pma1p isoforms (Ser800Ala, Glu803Gln) with increased in vitro H(+)/ATP stoichiometries was analysed by measuring biomass yields of anaerobic maltose-limited chemostat cultures expressing only the different PMA1 alleles. In vivo H(+)/ATP stoichiometries of wildtype Pma1p and the two isoforms did not differ significantly.

Energy coupling in Saccharomyces cerevisiae: selected opportunities for metabolic engineering.

Free-energy (ATP) conservation during product formation is crucial for the maximum product yield that can be obtained, but often overlooked in metabolic engineering strategies. Product pathways that do not yield ATP or even demand input of free energy (ATP) require an additional pathway to supply the ATP needed for product formation, cellular maintenance, and/or growth. On the other hand, product pathways with a high ATP yield may result in excess biomass formation at the expense of the product yield. This mini-review discusses the importance of the ATP yield for product formation and presents several opportunities for engineering free-energy (ATP) conservation, with a focus on sugar-based product formation by Saccharomyces cerevisiae. These engineering opportunities are not limited to the metabolic flexibility within S. cerevisiae itself, but also expression of heterologous reactions will be taken into account. As such, the diversity in microbial sugar uptake and phosphorylation mechanisms, carboxylation reactions, product export, and the flexibility of oxidative phosphorylation via the respiratory chain and H(+) -ATP synthase can be used to increase or decrease free-energy (ATP) conservation. For product pathways with a negative, zero or too high ATP yield, analysis and metabolic engineering of the ATP yield of product formation will provide a promising strategy to increase the product yield and simplify process conditions.

De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology.

Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.

Laboratory evolution of new lactate transporter genes in a jen1Δ mutant of Saccharomyces cerevisiae and their identification as ADY2 alleles by whole-genome resequencing and transcriptome analysis.

Laboratory evolution is a powerful approach in applied and fundamental yeast research, but complete elucidation of the molecular basis of evolved phenotypes remains a challenge. In this study, DNA microarray-based transcriptome analysis and whole-genome resequencing were used to investigate evolution of novel lactate transporters in Saccharomyces cerevisiae that can replace Jen1p, the only documented S. cerevisiae lactate transporter. To this end, a jen1Δ mutant was evolved for growth on lactate in serial batch cultures. Two independent evolution experiments yielded growth on lactate as sole carbon source (0.14 and 0.18 h(-1) , respectively). Transcriptome analysis did not provide leads, but whole-genome resequencing showed different single-nucleotide changes (C755G/Leu219Val and C655G/Ala252Gly) in the acetate transporter gene ADY2. Introduction of these ADY2 alleles in a jen1Δ ady2Δ strain enabled growth on lactate (0.14 h(-1) for Ady2p(Leu219Val) and 0.12 h(-1) for Ady2p(Ala252Gly) ), demonstrating that these alleles of ADY2 encode efficient lactate transporters. Depth of coverage of DNA sequencing, combined with karyotyping, gene deletions and diagnostic PCR, showed that an isochromosome III (c. 475 kb) with two additional copies of ADY2(C755G) had been formed via crossover between retrotransposons YCLWΔ15 and YCRCΔ6. The isochromosome formation shows how even short periods of selective pressure can cause substantial karyotype changes.

Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield.

Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-proton symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9% higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5' coding sequences of SUC2, resulting in predominant (94%) cytosolic localization of invertase. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 90 generations of laboratory evolution in anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11% higher ethanol yield on sucrose in chemostat cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. After deletion of both copies of the duplicated AGT1, growth characteristics reverted to that of the unevolved SUC2 and iSUC2 strains. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.

Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase.

Increasing free-energy conservation from the conversion of substrate into product is crucial for further development of many biotechnological processes. In theory, replacing the hydrolysis of disaccharides by a phosphorolytic cleavage reaction provides an opportunity to increase the ATP yield on the disaccharide. To test this concept, we first deleted the native maltose metabolism genes in Saccharomyces cerevisiae. The knockout strain showed no maltose-transport activity and a very low residual maltase activity (0.03 µmol mg protein(-1)min(-1)). Expression of a maltose phosphorylase gene from Lactobacillus sanfranciscensis and the MAL11 maltose-transporter gene resulted in relatively slow growth (µ(aerobic) 0.09 ± 0.03 h(-1)). Co-expression of Lactococcus lactis ß-phosphoglucomutase accelerated maltose utilization via this route (µ(aerobic) 0.21 ± 0.01 h(-1), µ(anaerobic) 0.10 ± 0.00 h(-1)). Replacing maltose hydrolysis with phosphorolysis increased the anaerobic biomass yield on maltose in anaerobic maltose-limited chemostat cultures by 26%, thus demonstrating the potential of phosphorolysis to improve the free-energy conservation of disaccharide metabolism in industrial microorganisms.