Yeast transcriptional device libraries enable precise synthesis of value-added chemicals from methanol

Abstract

Natural methylotrophs are attractive methanol utilization hosts, but lack flexible expression tools. In this study, we developed yeast transcriptional device libraries for precise synthesis of value-added chemicals from methanol. We synthesized transcriptional devices by fusing bacterial DNA-binding proteins (DBPs) with yeast transactivation domains, and linking bacterial binding sequences (BSs) with the yeast core promoter. Three DBP–BS pairs showed good activity when working with transactivation domains and the core promoter of PAOX1 in the methylotrophic yeast, Pichia pastoris. Fine-tuning of the tandem BSs, spacers and differentiated input promoters further enabled a constitutive transcriptional device library (cTRDL) composed of 126 transcriptional devices with an expression strength of 16–520% and an inducible TRDL (iTRDL) composed of 162 methanol-inducible transcriptional devices with an expression strength of 30–500%, compared with PAOX1. Selected devices from iTRDL were adapted to the dihydromonacolin L biosynthetic pathway by orthogonal experimental design, reaching 5.5-fold the production from the PAOX1-driven pathway. The full factorial design of the selected devices from the cTRDL was adapted to the downstream pathway of dihydromonacolin L to monacolin J. Monacolin J production from methanol reached 3.0-fold the production from the PAOX1-driven pathway. Our engineered toolsets ensured multilevel pathway control of chemical synthesis in methylotrophic yeasts.

Promoter engineering enables precise metabolic regulation towards efficient β-elemene production in Ogataea polymorpha

“…We also constructed two hybrid promoters with a two-fold increase in activities by identifying and integrating UAS regions. Tandem UAS region has been proved as an efficient strategy to increase the promoter strengths [ 54 , 55 ], but the increased leaky expression needs further exploration. Compared with other dynamic systems responsive to extracellular inducer like copper [ 56 ], estrogen [ 57 ], metabolites malonyl-CoA [ 58 , 59 ], and environmental factors like blue light [ 60 ] or temperature [ 61 ], the growth phase-dependent promoters we have identified are more convenient and inexpensive to use.…”

Transcriptome Identification and Analysis of Fatty Acid Desaturase Gene Expression at Different Temperatures in Tausonia pullulans 6A7

“…The significant enrichment of metabolic processes according to the GO annotation reflected the biological significance of yeast as a cell factory. Synthetic biology aimed at standardizing, modularizing, and innovating cellular functions has made great strides with the rapid advances in DNA synthesis and next-generation sequencing technologies [24]. Here, we review important advances in the synthetic biology of the brewer's yeast Saccharomyces cerevisiae, an important eukaryotic model and widely used cell factory [25].…”

Heterologous Single-Strand DNA-Annealing and Binding Protein Enhance CRISPR-Based Genome Editing Efficiency in Komagataella phaffii

“…Komagataella phaffii has been extensively employed in the industrial enzyme and biopharmaceutical industries as a stable and mature protein production platform; efficient genetic engineering tools are essential for achieving high titers of target proteins in K. phaffii. , Multiple genes must be engineered to optimize protein production including transcription factors that enhance transcription efficiency, chaperones that facilitate protein folding, proteases involved in target protein degradation, and genes associated with glycosylation. However, single-crossover recombination using resistance genes is insufficient for seamless gene disruption and integration of multiple genes.…”